Hirohara H, Philipp M, Bender M L
Biochemistry. 1977 Apr 19;16(8):1573-80. doi: 10.1021/bi00627a007.
The pH dependence for acylation of alpha-chymotrypsin by N-acetyltryptophan p-nitrophenyl-, p-nitrothiophenyl-, ethyl-, and thiolethyl esters has been studied by the stopped-flow technique. Values for the acylation rate constant, k2, and the binding constant, KS, were obtained by using measurements of phenolate release, for the p-nitrophenyl esters, and proflavin displacement, for the ethyl esters. The oxygen esters tested have slightly higher k2 values, and substantially higher KS values relative to the analogous thiol esters. Whereas k2/KS for the thiolethyl ester is higher than that for the analogous oxygen ester, the k2/KS values for oxy- and thio-p-nitrophenyl esters are nearly identical. These data are interpreted to indicate rate-determining formation of a tetrahedral intermediate in acylation of alpha-chymotrypsin by p-nitrophenyl esters, and rate-determining breakdown of such an intermediate in the case of the ethyl esters. It is also concluded that the oxygen to sulfur substitution causes a substantial increase in the proportion of nonproductive binding in these substrates. pH dependent k2 and KS values were used to calculate values for k1 and k-1, the binding and debinding rate constants for the two p-nitrophenyl compounds. This is the first such calculation based on experimentally determined acylation rate constants.
采用停流技术研究了N - 乙酰色氨酸对硝基苯酯、对硝基硫苯酯、乙酯和硫代乙酯对α-胰凝乳蛋白酶进行酰化反应时的pH依赖性。通过测量对硝基苯酯的酚盐释放以及乙酯的普罗黄素置换，获得了酰化速率常数k2和结合常数KS的值。相对于类似的硫醇酯，所测试的氧酯具有略高的k2值和显著更高的KS值。虽然硫代乙酯的k2/KS高于类似的氧酯，但氧代和硫代对硝基苯酯的k2/KS值几乎相同。这些数据被解释为表明对硝基苯酯酰化α-胰凝乳蛋白酶时四面体中间体的形成是速率决定步骤，而对于乙酯来说，这种中间体的分解是速率决定步骤。还得出结论，氧被硫取代导致这些底物中非生产性结合的比例大幅增加。利用pH依赖性的k2和KS值计算了两种对硝基苯化合物的结合速率常数k1和解离速率常数k-1的值。这是基于实验测定的酰化速率常数进行的首次此类计算。
